Methods and devices for marking a solid and subsequently detecting the markings

ABSTRACT

A method of marking a solid article or item comprises applying thereto beads having nucleic acids attached thereto. A method of monitoring an interaction between any material, article or item and a person or animal comprises providing a device capable of producing an aerosol containing a nucleic acid label and means to activate the aerosol on interference by a person or animal with the material, article or item. Methods of marking a material comprise adding to the material a plurality of beads having attached thereto a plurality of nucleic acid molecules. At least two distinct primer sequences are associated with different nucleic acid molecules.

This invention relates to a security device and a method which enablesan ultrasensitive microtrace to be used to demonstrate a directrelationship or interaction between any material, article or item(solid, liquid or gaseous) and man under appropriate conditions where itis required to demonstrate that a relationship or interaction occurred.In particular the security device will demonstrate that a givenindividual or individuals attempted to steal or did steal a materialarticle or item protected by the security device.

The present invention also relates more generally to methods of markinga material and subsequently detecting that the material has been markedand identifying the marker.

There is a widespread requirement to be able to trace the path taken bya given material as it moves from one location to another. The movementmay be of natural materials (e.g. the flow of water in sub-surfaceaquifers) or materials which have been processed or manufactured by man(e.g. any article constructed by man in a manufacturing process ornatural resources such as grains and minerals). In all these situationsthere may be reasons why it is necessary to develop specific proceduresto trace these movements. It may be that direct observation is notpossible, e.g., when following the path of a stream underground. It maybe that it is necessary to monitor the movement of goods without thedirect knowledge of the transporters or, for legal reasons, to provethat the appearance of a material at a particular point in the biospherewas due to at the same material originating from a known starting point.

For example, the articles of manufacture may be stolen in transit orresold at a much lower price than that set by the supplier by anunscrupulous distributor for example car boot sales. A key problem inbringing a conviction is identification of the particular articles sold,to establish that the goods have been stolen or resold from a particulardistributor. Problems also occur with liquids such as petroleum whichare routinely washed out of carriers into the sea. It is almostimpossible to identify which carrier has discharged the oil and as suchprosecutions and convictions for polluting the seas are rarely brought.

Further problems are associated with the movement of natural materials,for example the movement of grain. It is particularly difficult todistinguish one batch of such natural materials from another. In thecase of grain, problems occur in the European Community with the grainbeing moved across several different borders to collect a number of EUsubsidies for the same batch of grain. A method of marking the grainwhich may be readily detected is necessary to prevent such fraud.

Our co-pending patent application published under No. WO91/17265discloses in general terms ways in which materials may be labelled inparticular using a DNA molecule to label the material. Our co-pendingPCT application published 3 Mar. 1994 under No. WO94/04918, discloses amethod of marking a liquid in particular with two labels one of which isnot a nucleic acid tag and one of which comprises DNA molecules. In eachcase, the unique microtrace comprising DNA molecules is added to thematerial, the resulting material is sampled after movement thereof, andthe presence of the microtrace additive in the sample is detected,analyzed and decoded.

In a preferred aspect described, the material being monitored is aliquid hydrocarbon, such as oil, and the microtrace additive is designedsuch that it cannot be easily removed from the hydrocarbon by aqueouswashing. Various methods are proposed for ensuring that the DNAmicrotrace remains in the hydrocarbon rather than partitioning into theaqueous phase, including linking the DNA to hydrophobic beads typicallyof from 1 to 5 μm diameter, designed to be retained in hydrocarbons andnot the aqueous phase. A method of marking a liquid and subsequentlydetecting that the liquid has been marked is disclosed, which methodcomprises: adding to the liquid an additive comprising a plurality ofparticles in an amount no greater than 1 part by weight of particles per10⁶ parts by weight of liquid, the particles comprising signal means toaid their detection and not being visible in the liquid to the nakedeye; sampling a portion of the liquid containing the said additive;detecting the presence of particles in the sample, with the proviso thatthe said signal means does not consist solely of a nucleic acid tag.

The previous applications and uses of microtrace DNA labels areprimarily concerned with the labelling of liquids and in particular withhydrocarbons. There is no disclosure of how the labelling ofmanufactured or natural articles, in particular solid articles might beperformed.

In a first aspect of the invention there is provided a method of markinga solid article or item comprising applying to said article microbeadshaving nucleic acids attached thereto.

The previous inventions deal with the type of particles, their size,marker properties, methods of application and detection, as well asaspects of the unique DNA molecules used to provide the unique labels.In all these applications the use of the microtrace DNA labels involvesthe addition to the material of the label comprising a single DNAsequence for each material, which is subsequently detected after aperiod of time during which time the material may have been moved.Knowledge of the unique nature of the DNA molecule, in particular asecret but predetermined sequence of bases within the DNA microtracemolecule enables the origin and movement path of the labelled materialto be determined with complete accuracy.

A problem with the prior publications is the requirement that each DNAmust be essentially distinct from other DNAs to provide the requiredspecificity.

In a second aspect of the invention there is provided a method ofmarking a material comprising adding thereto a plurality of microbeadshaving attached thereto a plurality of nucleic acid molecules, at leasttwo distinct primers being associated with different nucleic acidmolecules.

The prior art uses of the microtrace DNA labels do not envisage thetransfer of the label from the material to any agent, especially man,who may legitimately or illegitimately be involved with the movement ofthe labelled material, article or item. There is also a need to be ableto protect any material, article or item by showing that an individualor individuals have been in contact with the labelled material, articleor item. This is particularly important in the case of any material,article or item which is being transported from one place to another andmay be a target for theft or damage. The invention will not prevent atheft occurring, except in the general sense of providing an additionaldeterrent which may cause potential thieves to desist, but will providemeans of uniquely and unequivocally linking a thief or potential thiefto the labelled material, article or item.

In a further aspect of the present invention, there is provided a methodof monitoring an interaction between any material, article or item and aperson or animal comprising providing a device adapted to produce anaerosol containing a nucleic acid label and means to activate saidaerosol on interference by a person or animal with the article, materialor item.

The general principle is to place a device on, within or close to thematerial, article or item to be protected. The device is capable ofgenerating an aerosol, upon a predetermined signal, to disperse amicrotrace DNA label sufficiently widely, rapidly and effectively to beable to-ensure that some of the DNA microtrace label is deposited on theagent, especially on an individual or individuals, which may beattempting to remove (steal) the material, article or item.

The microtrace aerosol may be a canister pressurised with a propellante.g. an aerosol product or compressed air and containing the DNAmicrotrace label, placed within a container, such as a suitcase orshipping container, only to be activated if the container is opened insome way by an individual who does not know how to disarm the microtraceaerosol prior to attempting to open the container. The act of openingthe container may in some way be used to trigger the microtrace aerosoland disperse the DNA microtrace into the atmosphere with sufficientforce that the individual opening the container will inevitably belayered with the microtrace. Subsequently samples from the individualexposed to the aerosol may be taken, for example, from skin or from thefibres of clothes, and the microtrace label analyzed.

The label may be in the form of a bead which has at least one nucleicacid molecule attached thereto and typically has between 100-4000nucleic acid molecules attached. The beads are typically formed of apolymeric material but may also be formed from natural materials such assilica and generally have a diameter within the range from 0.05 μm to100 μm, preferably within the range from 0.01 to 5 μm. Exemplarymicrobeads/spheres are commercially available from Dynal (UK) Ltd ofWirral, Merseyside, United Kingdom under the generic tradenames ofDYNABEADS and DYNASPHERES. The preparation of these beads is disclosedin e.g. European Patent Publication Nos. 91453, 10986 and 106873 andU.S. Pat. Nos. 4186120, 4530956, 4563510 and 4654267. The DNA isattached to the bead through biotin-streptavidin or neutralite bindingor through direct --COOH or --NH₂ mediated or other covalent links.

Additional features of the microtrace aerosol may aid the detection ofindividuals who have been exposed to the aerosol. For example additionalbeads having a fluorescent label might be included in the aerosol or thenucleic acid labelled beads may be fluorescently labelled and may bedetected in situ, e.g. on skin or clothes, as a visual signal generatedwhen appropriate light is shone on the beads to activate the fluorescentlabel. Alternatively a dye of some nature may be included in theaerosol, and the first stage of detection will be to define a region ofskin or clothing which shows the presence of the dye, providing initialevidence that this may be the region to examine more closely for thepresence of the microtrace label in order to find and isolate the DNAmicrotrace beads. Alternatively beads may be provided with half of aspecific binding pair e.g. antigen or antibody, may be radiolabelled,magnetic or include enzymes to said detection.

The device may be modified to provide systems for protecting property aspart of a house or business property security system such that in theevent of illegal entry the device may be activated to spray intruderswith a unique microtrace label which might facilitate the detection andunequivocal identification of the intruder. Another modification of thebasic device might be as a car alarm system that sprays intruders whoillegally enter a vehicle. In addition to the usage envisaged abovewhere the device is passively activated, for example, when a suitcase istampered with, the aerosol device may also be used knowingly by oneindividual who holds the device or is in a position to activate thedevice. For example, the microtrace aerosol might be used as the basisof a personal protection device to spray unique labels onto an attackeror potential attacker. The basic device may be modified to anycircumstances in which the transfer of the microtrace label by anaerosol to identify a second individual or material, article or itemmight be advantageous.

Retail articles or natural products to be labelled according to theinvention may be sprayed using an aerosol device or alternatively thebeads may be otherwise applied e.g. by painting on in liquid form or asuspension of beads incorporated in a pen for marking the article. Thebeads may be incorporated in an ink to be printed on the article orpackaging for the article. The beads may be applied to a small region ofthe article, identifiable by the marker or supplier so that noadditional label is required to detect the presence of the beads, butthe beads may be directly isolated from the relevant portion of thearticle for analysis. The method by which the beads are applied willvary depending on the article to be labelled. For example, grain couldbe sprayed with the beads at an appropriate point in the processing inthe same manner that the grain might be sprayed with fungicide whereas asmall region of a retail article could be labelled perhaps by applyingthe beads to a small area of the article using a pen to deposit the beadsuspension.

In each case, either the whole stock issuing from a particularmanufacturer might be labelled, so that each batch might be specificallytagged. Alternatively, batches being delivered to distributors who wereunder suspicion might be labelled, or random batches tagged to act as adeterrent to would-be fraudsters.

The beads may be suspended in solution for example an aqueous solutionbetween 10-100,000 beads per ml. Once the solution has dried, the beadsadhere to the surface to which they have been applied. Lacquers oradhesives may be incorporated into the solution to aid adhesion.Alternatively a layer of lacquer or adhesive may be applied over themicrobeads to protect them from wearing off.

The DNA coated microbeads are reasonably resilient. In the dry form, theDNA is not readily susceptible to the attack of nucleases and so, forexample, may successfully be used to label grain. Alternatively, the DNAmay be modified to increase resistance to nuclease attack. Given thatthe labels are often used to monitor retail items during transportationand initial sales, they do not come into contact with extremes of heatwhich otherwise might damage the DNA. DNA can withstand some UVexposure. While strong UV might cause damage, this would be pinpointedto one portion of the microbead only and DNA present in the remainder ofthe beads could still usefully be detected and analyzed.

The article, material or item bearing the DNA labelled beads may beanalyzed, firstly to detect whether DNA labelled beads are present andwhere they are located using, for example, the fluorescent or magneticproperty of the bead. The beads provide a focal point for the DNAallowing the DNA to be more readily analyzed and detected. Followinglocation of the beads, the beads may be washed out of or off thematerial or article or clothing of a person and the polymerase chainreaction (PCR) performed directly on the DNA labelled beads usingappropriate primers. The PCR may be carried out on DNA which has beendetached from the beads although it is preferable and more efficient tocarry out the PCR directly on the DNA labelled beads.

The microbeads may be prepared having at least two distinct DNAmolecules bound to each bead, the total number of DNA moleculespreferably lying within the range of 100-4000 molecules per bead. Thedistinct DNA molecules have separate primers for PCR, each primer beingselected from a different set of primers for example being one of tenknown primers. Once a bead has been isolated, the PCR reaction iscarried out using each of the primers from the first set and each of the10 primers from the second set. Successful PCR will indicate whichprimers are present. This allows for the same pool of DNA primers to beused to generate many easily identifiable DNA molecules and beads whichare distinguished from each other.

The invention is hereinafter described in more detail by way of exampleonly with reference to the accompanying drawings in which:

FIG. 1 is a schematic diagram of an aerosol device for dispersing DNAmicrotrace beads; and

FIG. 2 shows four different DNA molecules arranged pairwise on twodistinguishable beads.

An apparatus as shown schematically in FIG. 1 designed to produce asuitable aerosol when activated by an appropriate signal from thematerial, article or item to be protected. The canister contains asuitable propellant (1), e.g. compressed air, which when releasedgenerates the aerosol containing the DNA labelled microbeads and anyother material from the reservoir (2). The signal may be any electricalsignal, mechanical or other physical signal, light signal, magneticsignal, or any other suitable means to open the valve (3) to release thepropellent and enable the aerosol to be formed.

The apparatus shown schematically in FIG. 1 is based on a device usedfor spraying paint by model makers, a Badger Air Brush 100 powered byHumbrol Power pack compressed air. However, other known aerosol devicescould be adapted to incorporate the microtrace either for immediatespraying or in a reservoir for later activation or application.

The reservoir (1) contains the microbeads with attached DNA microtracemolecules in suspension. The microtrace beads may be in a gaseous orliquid phase at concentrations which give a high concentration of beadsin the aerosol. Typically the beads will be in a concentrated form suchthat when dispersed in the aerosol, 1 ml of aerosol contains between 10and 10⁸ beads, although higher and lower bead densities may be suitablefor particular applications. Normally the suspending phase will be waterbut any other liquid will serve as the dispersant for the aerosol.

The microtrace beads may follow any of the formulations previouslydescribed. For example, the beads Q-435 from Dyno Particles AS, P.O. Box160, N-2001, Lillestrom, Oslo, Norway (5.5 μm beads 89.5% polystyrene,5.5% divinylbenzene and 5% methylmethacrylate coated with streptavidinfor attachment of biotin labelled DNA oligonucleotides carrying theknown DNA base sequence of the microtrace label) may be used. Furtherthese or other beads labelled with a fluorescent chromaphore, such asCoumarin 6, might be used as an additional indicator of the presence ofthe microtrace beads from a collected sample. Beads such as DynoParticles beads MP-887 (0.5 μm diameter with Coumarin 6) or MP-821 (0.25μm diameter with Coumarin 6) might be used.

The reservoir may also contain separately from the beads additionalindicators for the presence of the dispersed aerosol. For example, thegaseous or liquid aerosol might also contain specific dyes, especiallyfluorescent dyes or other dyes detectable by a range of simple (light)or complex (electron microscopy, flow cytometry, NMR, IR or otherspectroscopic techniques) procedures. The purpose of these adjunctcomponents to the microtrace beads will be to provide convenientindicators that the aerosol spray has been deposited on an individualdirectly, his clothes or other items belonging to the tagged individual.Their purpose will be to indicate quickly and accurately what regionsshould be sampled in detail to investigate for the presence of themicrotrace beads. Because of the lack of variability in the possiblenumber of adjunct dyes or other indicators they will not in themselvesgive the level of uniqueness possible with the various DNAoligonucleotides used as the key microtrace labels. In addition theaerosol may contain additional material, for example a lacquer such asHumbrol nitrate cellulose dope (Hull UK) or adhesive, to aid theattachment of the microtrace beads to the intended target to ensure thatthe beads are layered onto and remain on the target individual ormaterial.

The beads are coated (attached either through the binding ofbiotin--contained within the DNA oligonucleotide--to either streptavidinor neutralite, or bound directly through some kind of --COOH or --NH₂ orother mediated covalent chemical link or any other method of securelyattaching the DNA molecules stably to the beads) with at least 100 DNAoligonucleotide molecules, typically 2000 molecules, having a knownunique label sequence would be suitable.

An aerosol may also be used to spray an article such as grain.

For the grain labelling procedure a mixture of fluorescent, DNA-labelled0.5 μm diameter beads (MP887 from Dyno) and fluorescent-labelled 4.5 μmdiameter beads (FC26 from Dyno) was used. The FC26 beads were selectedas suitable beads for rapid identification under UV microscopy. TheMP887 beads were used as the DNA carriers. For the two bead mixture inthe correct ratios a stock suspension of FC26 beads at 2.7×10⁷ beadsml⁻¹ in TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0) was prepared. 100μl stock FC26 bead suspension was added to 1 ml stock DNA-labelled MP887suspension. The two bead stock solution was diluted with TE buffer toyield a suspension containing 1×10⁶ FC26 beads ml⁻¹ and 1×10⁸DNA-labelled MP887 beads ml⁻¹. 1.0 ml diluted two bead suspension wassprayed onto 100 g wheat grain spread out on a tray and allowed to airdry for a few minutes. Labelled grain was stored in plastic containersat room temperature (ca 15° to 36° C.). Labelling of the grain was shownby epiflourescence microscopy using an excitation wavelength of 495 nmand an emission wavelength of 510 nm, and magnification ranging from x80 to x 800.

Samples of grain can be routinely analyzed using epifluorescence tocheck for the presence of fluorescent beads. A wide range of sampleshave been analyzed following various storage and treatments with 100%success in the case of samples which have been labelled using theseprocedures. Samples labelled for up to 9 months continue to show thepresence of beads with no indication of any significant changes in thenumber of beads expected to be observed.

5.0 g grain was selected and washed 3 times in 5.0 ml deionised water,and the washings pooled (15.0 ml). The washings were filtered through aseries of polycarbonate membrane filters (Nucleopore) in the sequence:

(a) 8.0 μm pore size to remove large particles including any significantgrain particles.

(b) 0.8 μm pore size to remove the next size of particles and FC26beads.

(c) 0.22 μm pore size to remove and concentrate the DNA labelled MP887beads.

Following each stage the membranes were washed with 5.0 ml deionisedwater to ensure that all the smaller particles not being retained by theparticular filter passed through the membrane. The final 0.22 μm filterwas carefully placed in a 1.5 ml plastic tube and 0.1 ml steriledeionised water added. The membrane and water was vortex mixed for 5.0min to remove the DNA-labelled beads from the membrane. The membrane wasremoved from the tube and the DNA labelled beads collected as a pelletfollowing centrifugation at 13,000 rpm for 20 min. 900 μl of thebead-free supernatant was carefully removed by pipette withoutdisturbing the bead containing pellet. The beads were then resuspendedin the remaining 100 μl sterile water by vigorous vortex mixing. 2.0 μlwas checked by epifluorescence microscopy to ensure that the 0.5 μmDNA-labelled MP887 beads had been recovered. The remaining suspensionwas stored at 0.4° C. for subsequent DNA amplification procedures.

In order to facilitate the construction of large numbers of unique DNAmicrotrace labels, more than one different DNA oligonucleotide may beattached to the beads. For example, each bead may carry two differentDNA molecules (FIG. 2). Moreover, each aerosol or additive may containmore than one recognisably different DNA-labelled bead. The beads mightbe distinguished on the basis of size, shape, surface architecture,chemical composition, chemical additives (e.g. different metal, dyes,fluorescent dyes, etc) or any other property which would enable a sampleto be analyzed for different types of beads.

The two different DNA molecules are recognised by having differentprimer sequences. A typical construction will be approximately 100 basepairs in length, the first and last 30 base pairs being primers for PCRamplification and the middle region comprising a specific DNA sequence.The specific DNA sequence may either be sequenced from the PCR primersor alternatively from an additional region known as the sequencingprimer. An additional sequence may be included between the PCR primer orsequencing primer and specific sequence to ensure that the entirespecific DNA sequence may be sequenced.

Where the two DNA molecules are used, either attached to the same orseparate beads, different PCR primers are used to distinguish themolecules from each other, the presence of a particular primer beingidentified by the amplification by PCR of the sequence in the presenceof a known primer. Table 7 shows how 3 DNA primers from a 3 dimensionalarray of primers might be selected for a particular bead.

Referring to table 7 below, two beads are exemplified, each having threeDNA molecules having 3 different primers. The first DNA molecule in eachcase has a primer selected from A-J, the second DNA molecule for eachbead has a primer selected from 1-10 and the third DNA molecule on eachbead has a primer selected from K to T. In this case the beads haveprimers C,2,M and E,9,N respectively.

                  TABLE 7                                                         ______________________________________                                             A     B                                                                  P1   1-    1-    C    D    E    F    G    H    I    J                         P3 P2                                                                              10    10    1-10 1-10 1-10 1-10 1-10 1-10 1-10 1-10                      ______________________________________                                        L                                                                             M                x                                                            N                          x                                                  O                                                                             P                                                                             Q                                                                             R                                                                             S                                                                             T                                                                             ______________________________________                                    

Once a bead has been isolated, a PCR may be carried out in the presenceof each of the primers A-J separately to discover which primer leads toamplification of the DNA indicating the primer present in the first DNAmolecule of the bead. Further amplification will identify which of theprimers 1 to 10 and K to T are present. If necessary the specific DNAsequence may then be elucidated to fully identify the bead.

The total number of useful unique DNA combinations for a given number ofDNA molecules (x) attached to one type of bead is shown in Table 1 andan arrangement matrix in Table 2. Table 3 shows the number of usefulunique pairwise bead combinations (p) for a given number of pairwise DNAcombinations on each bead (n) and an arrangement matrix in Table 4. Forexample, for 100 different DNA oligonucleotides and a microtrace productcontaining two different beads selected from 4 different beads, thetotal number of unique labels (73.477×10⁶) is shown in Table 5. Asimilar table for 200 different DNA molecules and the same number andcombination of beads shows that 182.9×10⁶ unique labels may be formed(Table 6). There is no limit to the number of unique microtrace labelssince the number of different DNA molecules can be increased or thenumber of beads in the aerosol or additive increased.

In the case of an aerosol following dispersal of the aerosol anddetection of a sample which has come into contact with the aerosol, thesample may be analyzed in the first instance simply to demonstrate thepresence (or otherwise) of the beads. Once beads have been detected,then the DNA may be analyzed as previously described by DNAamplification and DNA sequencing to determine the nature of the label.By this means conclusive links may be established between the nature ofan aerosol sample and the exact origin and position of the aerosol.

                                      TABLE 1                                     __________________________________________________________________________    Number of useful unique pairwise DNA combinations (n) for a given             number of DNA molecules (x) on one bead.                                      Total              Number of                                                                              Number                                            number of                                                                          Arrangement of                                                                              pairwise DNA                                                                           of times                                          DNA  useful unique                                                                        Number of                                                                            combinations                                                                           one DNA                                           molecules                                                                          pairwise DNA                                                                         pairwise DNA                                                                         by formula                                                                             molecule                                                                           % use of one                                 available                                                                          combinations                                                                         combinations                                                                         calculation                                                                            used DNA molecule                                 x    (see Table 2)                                                                        n      n = {x.sup.2 - x}/2                                                                    {x - 1}                                                                            {x  - 1}*100/n                               __________________________________________________________________________    2    ab     1      {4 - 2}/2 = 1                                                                          1    100                                          3    ab ac  3      {9 - 3}/2 = 3                                                                          2    67                                                bc                                                                       4    ab ac ad                                                                             6      {16 - 4}/2 = 6                                                                         3    50                                                bc bd                                                                         cd                                                                       5    ab ac ad ae                                                                          10     {25 - 5}/2 = 10                                                                        4    40                                                bc bd be                                                                      cd ce                                                                         de                                                                       6    ab . . . af                                                                          15     {36 - 6}12 = 15                                                                        5    33                                                bc . . . bf                                                                   cd . . . cf                                                                   de . . . df                                                                   ef                                                                       100  etc    etc    {10000 - 100}/2 =                                                                      99   2                                                               4,950                                                      200  etc    etc    {40000 - 200}/2 =                                                                      199  1                                                               19,900                                                     1000 etc    etc    499,500  999  0.2                                          1 × 10.sup.18                                                                etc    etc    5 × 10.sup.35                                                                    1 × 10.sup.18                                                                2 × 10.sup.-18                         __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        Arrangement of useful unique pairwise DNA combinations (n)                    for a given number of DNA molecules (x) on one bead.                          ______________________________________                                         ##STR1##                                                                     ______________________________________                                         ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                     See Table 1 for calculations or values of n for different values of x     

    TABLE 3                                                                       __________________________________________________________________________    Number of useful unique pairwise bead combinations (p) for a given            number of pairwise DNA combinations on each bead (n).                                                 Number of                                                         Number of useful, unique                                                                  times one                                                         pairwise bead                                                                             DNA                                                   Total                                                                              Number of                                                                            combinations with two                                                                     molecule                                              number of                                                                          pairwise DNA                                                                         DNA molecules per bead                                                                    used in total                                                                       % use of one DNA                                DNA  combinations                                                                         by formula calculation                                                                    combinations                                                                        molecule                                        molecules                                                                          (see Table 1)                                                                        (see Table 4)                                                                             {(x - 1).sup.2 -                                                                    {{(x - 1).sup.2 - (x - 1)}/                     x    n      p = {n.sup.2 - 2n}/2                                                                      (x - 1)}/2                                                                          2}*100/p                                        __________________________________________________________________________    100  4950   {24,502,500 - 9900}/2 =                                                                   4851  0.04                                                        12,246,300                                                        200  19,900 {396,010,000 - 39800}/2 =                                                                 19,701                                                                              0.01                                                        197,985,100                                                       1000 499,500                                                                              1.247 × 10.sup.11                                                                   498,501                                                                             0.0004                                          1 × 10.sup.18                                                                5 × 10.sup.35                                                                  1.25 × 10.sup.71                                                                    5 × 10.sup.35                                                                 4.0 × 10.sup.-34                          __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                        Number of useful unique pairwise bead combinations (p) for a                  given number of pairwise DNA combinations on each bead (n).                   ______________________________________                                         ##STR5##                                                                     ______________________________________                                         ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                     See Table 3 for calculations on values of p for different values of n     

    TABLE 5                                                                       ______________________________________                                        Number of useful unique pairwise DNA from 100 different DNA                   molecules on 2 different beads selected from 4 different                      ______________________________________                                        beads.                                                                         ##STR9##                                                                     ______________________________________                                         ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                

For 100 different DNA molecules arranged in different pairwisecombinations on a mixture of two different beads taken from fourdifferent types of bead there are:

                  TABLE 6                                                         ______________________________________                                        Number of useful unique pairwise combinations of DNA molecules                from 200 different DNA molecules on 2 a pairwise lead combination             selected from 4 different beads.                                              ______________________________________                                         ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                

For 200 different DNA molecules arranged in different pairwisecombinations on a mixture of two different beads taken from fourdifferent types of bead there are:

We claim:
 1. A method of marking a solid and subsequently detecting thatthe solid has been marked, said method comprising:adding to a liquid anadditive comprising a plurality of microbeads in an amount no greaterthan 1 part weight of microbeads per 10⁶ parts weight liquid, saidplurality of microbeads comprising two or more signal means to aid theirdetection and code means to aid identification, said microbeads notbeing visible in the liquid to the naked eye; said additive comprisingeither (a) two or more microbeads, each microbead having differentsignal means, and at least one microbead having a code means or (b) amicrobead having two or more different signal means and at least onecode means; said code means and one of said signal means comprising anucleic acid and another of said signal means comprising a non-nucleicacid signal means; applying said liquid to said solid and allowing saidliquid to dry to mark the solid; detecting the presence on the solid ofsaid microbeads having said non-nucleic acid signal means; sampling thesolid marked with said additive; and decoding said code means, therebydetecting that the solid had been marked and identifying the solid.
 2. Amethod as claimed in claim 1 wherein the particles are applied byspraying said solid with liquid containing said microbeads.
 3. A methodas claimed in claim 1 wherein between 100-10⁸ microbeads are present perml of liquid.
 4. A method according to claim 1 wherein said liquidadditionally comprises a lacquer or adhesive.
 5. A method according toclaim 1 further comprising the step of applying a lacquer or adhesiveover said microbeads on said solid.
 6. A method according to claim 1wherein said second signal means comprises a fluorescent dye.
 7. Amethod according to claim 1 in which the solid is selected from thegroup consisting of an article of manufacture, naturally occurringmaterial, humans and animals.
 8. A method as claimed in claim 1, whereinat least two distinct primer sequences are associated with said nucleicacid signal means.
 9. A method according to claim 8 wherein saidmicrobeads have an average size from 0.05 to 5 μm.
 10. A methodaccording to claim 8 wherein each said microbead has between 100 and2000 nucleic acid molecules attached thereto.
 11. A method according toclaim 8 wherein said two distinct primer sequences are attached to thesame microbead.
 12. A method of monitoring an interaction between anymaterial, article, or item and a person or animal, comprising:adding toa liquid an additive comprising a plurality of microbeads in an amountno greater than 1 part weight of microbeads per 10⁶ parts weight liquid,said plurality of microbeads comprising two or more signal means to aidtheir detection and code means to aid identification, said microbeadsnot being visible in the liquid to the naked eye; said additivecomprising either (a) two or more microbeads, each microbead havingdifferent signal means, and at least one microbead having a code meansor (b) a microbead having two or more different signal means and atleast one code means; said code means and one of said signal meanscomprising a nucleic acid and another of said signal means comprising anon-nucleic acid signal means; providing a device adapted to produce anaerosol containing the liquid and the additive and means to dispersesaid aerosol during said interaction; and detecting the presence on saidperson, animal, material, article, or item of said microbeads havingsaid non-nucleic acid signal means; detecting the presence on saidperson, animal, material, article, or item of said microbeads havingsaid signal means comprising a nucleic acid; and decoding said codemeans, thereby detecting that the person, animal, material, article, oritem had been marked by interaction between said material, article, oritem and said person or animal.